The present invention relates to chemical compounds, in particular 1,4-diazacycloheptanes, to processes for their preparation and to chemical intermediates useful in such processes. The present invention further relates to 1,4-diazacycloheptanes, to pharmaceutical compositions containing them and to their use in methods of therapeutic treatment of animals including man, in particular in the treatment of neurological disorders.
Neurological disorders, for which the present compounds are useful, include stroke, head trauma, transient cerebral ischemic attack, and chronic neurodegenerative disorders such as Alzheimer""s disease, Parkinson""s disease, diabetic neuropathy, amyotrophic lateral sclerosis, multiple sclerosis, vascular dementia and AIDS-related dementia.
The compounds useful in the present invention are believed to act by binding with the [3H]-emopamil binding site. Emopamil has classically been thought of as a neuroprotective agent whose efficacy is most likely derived from actions at either voltage-sensitive calcium channels (VSCC) or 5-HT2 receptors. An apparent paradox to this logic is that verapamil, although chemically and pharmacologically very similar to emopamil, is not neuroprotective. While the lack of neuroprotective efficacy by verapamil was initially explained by lack of CNS penetration, recent studies suggest other factors may be involved (Keith et al., Br. J. Pharmacol. 113: 379-384, 1994).
[3H]-Emopamil binding defines a unique high affinity site that is not related to VSCC, is found in the brain, but is most prevalent in the liver (Moebius et al., Mol. Pharmacol. 43: 139-148, 1993). Moebius et al. have termed this the xe2x80x9canti-ischemicxe2x80x9d binding site on the basis of high affinity displacement by several chemically disparate neuroprotective agents. In liver, the [3H]-emopamil binding site is localized to the endoplasmic reticulum.
Neuroprotective compounds are known, for example emopamil and ifenprodil, that exhibit high affinity for the [3H]-emopamil binding site. However these are not selective inhibitors and exhibit activity either at neuronal VSCC, the polyamine site of the NMDA receptor (N-Methyl-D-aspartate) and/or the sigma-1 binding site. We have now found a class of compounds that show selective action at the [3H]-emopamil binding site that are neuroprotective in global and focal models of cerebral ischemia without acting directly at either VSCC or NMDA receptors, and consequently exhibit fewer associated side effects than are conventionally seen with either emopamil (hypotension) or ifenprodil (behavioural manifestations). Such compounds are especially useful in treating neurodegeneration resulting from ischemia, for example in Alzheimer""s disease, vascular dementia, Parkinson""s disease, Huntington""s disease and AIDS-related dementia. In another aspect such compounds are especially useful in treating stroke as they provide neuronal protection by preventing neuronal death in the penumbra region surrounding the core infarct. Accordingly the present invention provides the use of a compound which binds selectively to the [3H]-emopamil binding site for treating neurodegeneration resulting from ischemia.
Accordingly the present invention provides a compound of the formula (I): 
wherein:
R is hydrogen, C1-10alkyl, C3-8cycloalkyl, C3-8cycloalkylC1-6alkyl, phenylC1-6alkyl or phenyl;
R1 is C1-6alkyl, C2-6alkenyl, C1-6alkoxy, halo, hydroxy, C1-6alkanoyl, haloC1-6alkyl, cyano or nitro;
m is 0, 1 or 2;
R2 is C1-6alkyl;
n is 1, 1 or 2;
wherein any phenyl ring is optionally substituted; p1 or a pharmaceutically acceptable salt or in vivo hydrolysable ester, amide or carbanate thereof
Any phenyl ring in R may be optionally substituted, for example by up to five substituents, preferably up to three substituents which may be the same or different. Typical substituents include: hydroxy; C1-6alkoxy for example methoxy; mercapto; C1-6alkylthio for example methylthio; amino; C1-6alkylamino for example methylamino; di-(C1-6alkyl)amino for example dimethylamino; carboxy; carbamoyl; C1-6alkylcarbamoyl for example methylcarbamoyl; di-C1-6alkylcarbamoyl for example dimethylcarbamoyl; C1-6alkylsulphonyl for example methylsulphonyl; arylsulphonyl for example phenylsulphonyl; C1-6alkylaminosulphonyl for example methylaminosulphonyl; di-(C1-6alkyl)aminosulphonyl for example dimethylamino-sulphonyl; nitro; cyano; cyano-C1-6alkyl for example cyanomethyl; hydroxyC1-6alkyl for example hydroxymethyl; amino-C1-6alkyl for example aminoethyl; C1-6alkanoyl-amino for example acetamido; C1-6alkoxycarbonylamino for example methoxycarbonylamino; C1-6alkanoyl for example acetyl; C1-6alkanoyloxy for example acetoxy; C1-6alkyl for example methyl, ethyl, isopropyl or tert-butyl; halo for example fluoro, chloro or bromo; trifluoromethyl or trifluoromethoxy. In another aspect a further typical substituent for any phenyl group is phenylC1-6alkoxy.
In one aspect the present invention provides a compound of the formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable amide or carbamate thereof, wherein
R is hydrogen, C1-6alkyl, C3-8cycloalkyl, C3-8cycloalkylC1-6alkyl, phenylC1-6alkyl or phenyl;
R1 is
C1-6alkyl, C1-6alkoxy, halo, hydroxy, haloC1-6alkyl, cyano or nitro; m is 0, 1 or 2; R2 C1-6alkyl; and n is 0, 1 or 2; wherein any phenyl ring is optionally substituted.
Suitably R is hydrogen; C1-10alkyl for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl (n-pentyl or 3-methylbutyl) or 2-ethylheptyl; C3-8cycloalkyl for example cyclopropyl, cyclobutyl or cyclopentyl; C3-8cycloalkylC1-6alkyl for example cyclopropylmethyl, cyclobutylmethyl or cyclopentylmethyl; phenylC1-6alkyl for example benzyl, 2-phenethyl or 3-phenylpropyl.
Favourably R is hydrogen or C1-6alkyl. In particular R is hydrogen or C1-4alkyl such as methyl, ethyl, isopropyl, n-propyl, n-butyl or isobutyl. In particular also R is C5-6alkyl for example 3-methylbutyl. Another particular value for R is phenylC1-6alkyl for example benzyl, 2-phenethyl or 3-phenylpropyl. In one aspect R is C1-6alkyl or phenylC1-6alkyl. Preferably R is methyl, 3-methylbutyl or 3-phenylpropyl. In one aspect R is C1-6alkyl or phenylC1-6alkyl.
Suitably R1 is C1-6alkyl for example methyl, ethyl or propyl; C2-6alkenyl for example vinyl; C1-6alkoxy for example methoxy, ethoxy or propoxy; halo for example bromo, chloro or fluoro; hydroxy; C1-6alkanoyl for example formyl or acetyl; haloC1-6alkyl for example trifluoromethyl; cyano or nitro
In one aspect R1 is C1-6alkyl, C1-6alkoxy, halo, hydroxy, haloC1-6alkyl, cyano or nitro.
Preferably R1 is C1-6alkoxy for example methoxy or ethoxy or is halo for example bromo chloro or fluoro. In a particularly preferred aspect, m is one and R1 is methoxy, for example at the 5-position or the 7-position of the 1,2,3,4-tetrahydronaphthalene ring system, most preferably at the 5-position. In another particularly preferred aspect, m is one and R1 is bromo or fluoro, for example at the 6-position of the 1,2,3,4-tetrahydronaphthalene ring system.
In another preferred aspect m is zero.
Suitably R2 is C1-6alkyl for example methyl or ethyl.
In a preferred aspect n is zero.
A particular class of preferred compounds is that of the formula (II): 
wherein R3 is hydrogen, or C1-6alkyl or pheny C1-6alkyl and R4 is hydrogen or C1-6alkoxy. In one aspect R3 is C1-6alkyl. In particular in the compounds of the formula (II), R4 is hydrogen and R3 is methyl, 3-methylbutyl or 3-phenylpropyl.
Particular compounds of the present invention include those of the Examples hereinafter; 4-(7-methoxy-1,2,3,4-tetrahydro-1-naphthalenyl)homopiperazine 1-methyl-4-(7-methoxy-1,2,3,4-tetrahydro-1-naphthalenyl)homopiperazine and 1-isopropyl-4-(5-methoxy-1,2,3,4-tetrahydro-1-naphthalenyl)homopiperazine
The compounds of the present invention possess a chiral centre at the 1-position of the 1,2,3,4-tetrahydronaphthalene ring system (that is the carbon atom to which the nitrogen containing ring is attached). Other chiral centres may be present when n is one or two and in any of the substituents R-R4.
The present invention covers all enantiomers, diastereoisomers and mixtures thereof of the compound of the formula (I) that inhibit the [3H]-emopamil binding site.
As mentioned hereinabove, the compounds of the present invention possess a chiral centre at the 1-position of the 1,2,3,4-tetrahydronaphthalene ring system. It is preferred that this centre has the S-stereochemistry under the Cahn-Prelog-Ingold sequence rules. It is preferred that any R or S-enantiomer is substantially free of the corresponding S or R-enantiomer, suitably 90%, more suitably 95%, and for example 96%, 97%, 98% or 99% free of the other enantiomer.
Suitable pharmaceutically acceptable salts include acid addition salts such as hydrochloride, hydrobromide, citrate and maleate salts and salts formed with phosphoric and sulphuric acid. In another aspect suitable salts are base salts such as an alkali metal salt for example sodium or potassium, an alkaline earth metal salt for example calcium or magnesium, or organic amine salt for example triethylamine.
In vivo hydrolysable esters, amides and carbamates hydrolyse in the human body to produce the parent compound. Such esters, amides and carbamates can be identified by administering, for example intravenously to a test animal, the compound under test and subsequently examining the test animal""s body fluids. Suitable in vivo hydrolysable groups include N-carbomethoxy and N-acetyl.
In order to use a compound of the formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester amide or carbamate thereof for the therapeutic treatment (including prophylactic treatment) of mammals including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
Therefore in another aspect the present invention provides a pharmaceutical composition which comprises a compound of the formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester amide or carbamate and a pharmaceutically acceptable carrier.
The pharmaceutical compositions of this invention may be administered in standard manner for the disease condition that it is desired to treat, for example by oral, topical, parenteral, buccal, nasal, vaginal or rectal administration or by inhalation. For these purposes the compounds of this invention may be formulated by means known in the art into the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation, and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions. A preferred route of administration is intravenously in sterile isotonic solution.
In addition to the compounds of the present invention the pharmaceutical composition of this invention may also contain, or be co-administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more disease conditions referred to hereinabove.
The pharmaceutical compositions of this invention will normally be administered to humans so that, for example, a daily dose of 0.05 to 75 mg/kg body weight (and preferably of 0.1 to 30 mg/kg body weight) is received. This daily dose may be given in divided doses as necessary, the precise amount of the compound received and the route of administration depending on the weight, age and sex of the patient being treated and on the particular disease condition being treated according to principles known in the art.
Typically unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention.
Therefore in a further aspect, the present invention provides a compound of the formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester, amide or carbamate thereof for use in a method of therapeutic treatment of the human or animal body.
In yet a further aspect the present invention provides a method of treating a disease condition wherein inhibition of the [3H]-emopamil binding site is beneficial which comprises administering to a warm-blooded animal an effective amount of a compound of the formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester, amide or carbamate thereof. The present invention also provides the use of a compound of the formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester, amide or carbamate thereof in the preparation of a medicament for use in a disease condition.
In another aspect the present invention provides a process for preparing a compound of the formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester, amide or carbamate thereof which process comprises:
a) reacting a compound of the formula (III) with a compound of the formula (IV): 
wherein R, R1, R2, m and n are as hereinbefore defined and L is a leaving group; or
b) deprotecting a compound of the formula (V): 
wherein R1, R2, m and n are as hereinbefore defined and P is a protecting group for R;
wherein any functional group is protected, if necessary, and:
i) removing any protecting groups;
ii) optionally converting a compound of the formula (I) into another compound of the formula (I);
iii) optionally forming a pharmaceutically acceptable salt or an in vivo hydrolysable ester, amide or carbamate.
Protecting groups may in general be chosen from any of the groups described in the literature or known to the skilled; chemist as appropriate for the protection of the group in question, and may be introduced by conventional methods.
Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule.
Specific examples of protecting groups are given below for the sake of convenience, in which xe2x80x9clowerxe2x80x9d signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.
A carboxyl protecting group may be the residue of an ester-forming aliphatic or araliphatic alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).
Examples of carboxy protecting groups include straight or branched chain (1-12C)alkyl groups (eg isopropyl, t-butyl); lower alkoxy lower alkyl groups (eg methoxymethyl, ethoxymethyl, isobutoxymethyl); lower aliphatic acyloxy lower alkyl groups, (eg acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl); lower alkoxycarbonyloxy lower alkyl groups (eg 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl); aryl lower alkyl groups (eg benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, benzhydryl and phthalidyl); tri(lower alkyl)silyl groups (eg trimethylsilyl and t-butyldimethylsilyl); tri(lower alkyl)silyl lower alkyl groups (eg trimethylsilylethyl); and (2-6C)alkenyl groups (eg allyl and vinylethyl).
Methods particularly appropriate for the removal of carboxyl protecting groups include for example acid-, base-, metal- or enzymically-catalysed hydrolysis.
Examples of hydroxyl protecting groups include lower alkyl groups (eg t-butyl), lower alkenyl groups (eg allyl); lower alkanoyl groups (eg acetyl); lower alkoxycarbonyl groups (eg t-butoxycarbonyl); lower alkenyloxycarbonyl groups (eg allyloxycarbonyl); aryl lower alkoxycarbonyl groups (eg benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri(lower alkyl)silyl (eg trimethylsilyl, t-butyldimethylsilyl) and aryl lower alkyl (eg benzyl) groups.
Examples of amino protecting groups include formyl, aralkyl groups (eg benzyl and substituted benzyl, p-methoxybenzyl, nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-p-anisylmethyyl and furylmethyl groups; lower alkoxycarbonyl (eg t-butoxycarbonyl); lower alkenyloxycarbonyl (eg allyloxycarbonyl); aryl lower alkoxycarbonyl groups (eg benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); trialkylsilyl (eg trimethylsilyl and t-butyldimethylsilyl); alkylidene (eg methylidene); benzylidene and substituted benzylidene groups.
Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis, for groups such as p-nitrobenzyloxycarbonyl, hydrogenation and for groups such as o-nitrobenzyloxycarbonyl, photolytically.
Pharmaceutically acceptable salts of the compound of the formula (I) may be prepared in any conventional manner for example from the free base and acid. In vivo hydrolysable esters, amides and carbamates may be prepared in any conventional manner.
The reaction between the compounds of the formulae (III) and (IV) is performed in conventional manner. Typically this reaction takes place in organic solvent for example an anhydrous aprotic solvent such as dimethylformamide, dimethylacetamide or tetrahydrofuran. The reaction is generally performed in the presence of a catalyst, such as an iodide salt for example potassium iodide, and is generally performed at ambient. or elevated temperature for example 0xc2x0-100xc2x0 C., more preferably 40xc2x0-80xc2x0 C.
In the compounds of the formula (III), L is a conventional leaving group such as halo for example chloro, iodo or bromo; or a tosylate for example p-toluenesulphonyloxy or methanesulphonyloxy.
In the compounds of the formula (III), the leaving group L may also represent oxo (xe2x95x90O), forming an a-tetralone ring system. Such compounds may be reacted with a compound of the formula (IV) under conventional conditions for reductive amination. Suitable conditions include the presence of a reducing agent such as hydrogen and a hydrogenation catalyst (for example palladium on carbon), or zinc and hydrochloric acid, or sodium cyanoborohydride, or sodium triacetoxyborohydride, or sodium borohydride, iron pentacarbonyl and alcoholic potassium hydroxide, or borane and pyridine or formic acid. The reaction is preferably carried out in the presence of a suitable solvent such as an alcohol, for example methanol or ethanol, and at a temperature in the range of 0-50xc2x0 C., preferably at or near room temperature.
The compounds of the formula (III) are either known or may be prepared in conventional manner as known to the organic chemist skilled in the art. One convenient manner is to convert the corresponding 1-hydroxy-1,2,3,4-tetrahydronaphthalene to the compound of the formula (III); for example by treating with thionyl chloride in the presence of pyridine to prepare the compound of the formula (III) wherein L is chloro.
Compounds of the formula (V) wherein P is a protecting group convertible to R may be deprotected in standard manner. Any suitable N-protecting group may be used and deprotected in conventional manner. Favourably P is C1-6alkoxycarbonyl and such compounds may be converted to compounds of the formula (I) wherein R is methyl for example by treating with a reducing agent such as lithium aluminium hydride. Certain compounds of the formula (V) are also in vivo hydrolysable esters, amides or carbamates of the compounds of the formula (I).
Compounds of the formula (I) wherein R is hydrogen may be converted to compounds of the formula (I) wherein R is other than hydrogen. For example such conversion may comprise conventional methods of alkylation with an appropriate alkylating agent or reductive amination. For example an isopropyl group may be prepared by reacting a compound of the formula (I) wherein R is hydrogen with acetone in the presence of a reducing agent such as sodium borohydride or sodium cyanoborohydride. A 2-methylpropyl group may be prepared by reacting a compound of the formula (I) wherein R is hydrogen with isobutyric acid in the presence of a reducing agent such as sodium borohydride or sodium cyanoborohydride.
Thus in another aspect the present invention provides a process for preparing a compound of the formula (I) wherein R is not hydrogen, especially where R is C1-10alkyl, from a compound of the formula (I) wherein R is hydrogen by reaction with an alkylating agent or by reductive amination.
As mentioned hereinabove, the compounds of the present invention possess a chiral centre at the 1-position of the 1,2,3,4-tetrahydronaphthalene ring system and the present invention encompasses the racemate and individual enantiomers. Enantiomers of the compound of the formula (I) may be prepared in conventional manner by resolution of a racemic compound. Alternatively enantiomers of the compounds of the formula (I) may be prepared in analogous manner to the racemates commencing with chiral starting-materials. In yet a further alternative, a chemical intermediate, for example of the formula (III), or the corresponding hydroxy compound, or of the formula (V), may be resolved and subsequently reacted without destroying chirality.
The following biological test methods, data and Examples serve to illustrate the present invention.
3H-Emopamil Binding to Guinea Pig Liver Membranes
The method of (xe2x88x92)-3H-emopamil binding was a modification of Zech, C., Staudinger R., Mxc3xchlbacher, J. and Glossmann, H. Novel sites for phenylalkylamines: characterization of a sodium-sensitive drug receptor with (xe2x88x92)-3H-emopamil. Eur. J. Pharm. 208: 119-130, 1991.
The reaction mixture contained:
Assay buffer: 10 mM Tris-HCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2% bovine serum albumin (BSA), pH 7.4 at 4xc2x0 C.
Radioligand: 0.96 nM (xe2x88x92)-3H-emopamil (Amersham).
Guinea pig liver membranes: 40 mg/mL original wet weight.
Compounds: 1-300 nM.
Total volume: 500 xcexcL.
This mixture was incubated for 60 minutes at 37xc2x0 C. The incubation was terminated by filtering with a Brandel Cell Harvester over Whatman GF/C filters that had been soaked for at least 120 minutes in 0.3% polyethylenimine (PEI) and washed three times with 5 mL of wash buffer containing 10 mM Tris-HCl, 10 mM MgCl2, 0.2% BSA, pH 7.4 at 25xc2x0 C. Specific binding was defined with 10 xcexcM emopamil. In general compounds with an IC50 Specific binding was defined with 10 xcexcM emopamil. In general compounds with an IC50 below 300 nM in this test were of interest and for example the compound of Example 4 gave a value of 17 nM.
Guinea-pig liver membrane preparation: Male guinea pigs were sacrificed by CO2 asphyxiation with dry ice. The livers were quickly excised and weighed and rinsed in membrane preparation buffer containing 10 mM Hepes, 1 mM Tris base-EDTA, 250 mM driven Teflon-glass homogenizer with three strokes on ice. The homogenate was centrifuged driven Teflon-glass homogenizer with three strokes on ice. The homogenate was centrifuged at 1000xc3x97g in a SS34 rotor for 5 minutes at 4xc2x0 C. The supernatant was filtered through 4 layers of gauze and then centrifuged at 8000xc3x97g for 10 minutes at 4xc2x0 C. This resulting supernatant was centrifuged at 40,000xc3x97g for 15 minutes at 4xc2x0 C. The resulting pellet was resuspended in assay buffer and centrifuged again at 40,000xc3x97g for 15 minutes at 4xc2x0 C. This pellet was resuspended in assay buffer (2.5 fold with respect to original wet weight) and homogenized with one stroke with the Teflon-glass homogenizer. Aliquots of 1 mL were stored at xe2x88x9270xc2x0 C.
3H-D-888 Binding to Rat Brain Cortical Membranes
The method of 3H-D-888 binding was a modification of Reynolds, I. J., Snowman, A. M. and Synder, S. H. (xe2x88x92)-[3H] Desmethoxyverapamil labels multiple calcium channel modular receptors in brain and skeletal muscle membranes: differentiation by temperature and dihydropyridines. J. Pharmacol. Exp. Ther. 237: no.3, 731-738, 1986.
The assay tubes contained the following:
assay buffer: 50 mM Hepes, 0.2% BSA, pH 7.4
radioligand: 1 xcfx80M 3H-D888 (Amersham)
rat cortical membranes: 6 mg/ml original wet weight
compounds: 0.3-100 xcexcM
Total volume: 1000 xcexcL
This mixture was incubated for 60 minutes at 25xc2x0 C. The assay was terminated by filtering with a Brandel Cell Harvester over Whatman GF/C filters that had been soaked for at least 120 minutes in 0.3% polyethylenamine (PEI) and washed three times with 5 mL of wash buffer containing 20 mM Hepes, 20 mM MgCl2, pH 7.4. Specific binding was measured with 10 xcexcM methoxyverapamil (D-600). This assay was used to determine in vitro selectivity of compounds vs. L-type voltage sensitive calcium channels, i.e high affinity for the 3H-D888 binding site would show a lack of selectivity. For example the compound of Example 4 gave a value of about 19.000 nM in this test.
Rat brain cortical membrane preparation: Male Sprague-Dawley Rats were sacrificed by decapitation and the brains were quickly excised. The cerebellum and brain stem were removed and discarded; and the rest of the brain was rinsed in 320 mM sucrose. The brain was then homogenized in a 10-fold volume of 320mM sucrose with a motor driven Teflon-glass homogenizer using 10 strokes on ice. The homogenate was spun at 1000xc3x97g for 10 glass homogenizer using 10 strokes on ice. The homogenate was spun at 1000xc3x97g for 10 minutes at 4xc2x0 C. in a SS-34 rotor. The supernatant was then spun at 29,000xc3x97g for 20 minutes. The resulting pellet was resuspended in membrane buffer (5 mM Hepes, 0.2% BSA, pH 7.4 ) to a final concentration of 60 mg original wet weight/mL.
Gerbil Global Model of Cerebral Ischemia
Male Mongolian gerbils (Charles River) weighing 60-70 grams are used in these experiments. They are housed in individual cages with food (Purina Rodent Chow) and water available ad libitum. The animal room is maintained at 23xc2x0 C.xc2x12xc2x0, and is on an automatic 12 hour light cycle.
The gerbils are brought to the surgical suite and dosed intraperitoneally with the test agent or vehicle, forty five minutes prior to surgery. Drugs are administered at a volume of 5 ml/kg (intraperitoneal). Vehicle is generally saline, with sodium phosphate added to adjust pH, if needed. Forty-five minutes after dosing the gerbils are anesthetized with halothane (3.3%) which is delivered along with oxygen (1.5 L/M) through a face mask. After the gerbils are anesthetized, halothane is continued at a maintenance level of 1.5-2% along with oxygen. The ventral surface of the neck is shaved and cleaned with alcohol. Surgical procedures are carried out on a thermostat-controlled heating pad set to 37xc2x0 C. An incision is made in the neck, the carotid arteries are dissected away from the surrounding tissue, and isolated with a 5 cm length of Silastic tubing. When both arteries have been isolated they are clamped with microaneurysm clips (Roboz Instruments). The arteries are visually inspected to determine that the blood flow has been stopped. After 5 minutes the clips are gently removed from the arteries and blood flow begins again. A sham control group is treated identically but is not subjected to carotid artery occlusion. The incisions are closed with suture and the gerbils removed from the anesthesia masks and placed on another heating pad to recover from the anesthesia. When they have regained the righting reflex and are beginning to walk around, they are again dosed with the test compound and returned to their home cages. This occurs approximately five minutes after the end of surgery.
Twenty-four hours post ischemia gerbils are tested for spontaneous locomotor activity, using a Photobeam Activity System from San Diego Instruments. They are individually placed in Plexiglas chambers measuring 27.5 cmxc3x9727.5 cmxc3x9715 cm deep. The chambers are surrounded by photocells, and every time a beam is broken one count is recorded. Each gerbil is tested for two hours, and cumulative counts are recorded at 30, 60, 90, and 120 minutes. Mean counts are recorded for each group and drug groups are compared to control with an ANOVA and Bonferroni post test. After each gerbil is tested it is returned to its home cage. At this time gerbils are also observed for any changes from normal behavior.
For the next two days no specific testing is performed, but the gerbils are observed two to three times per day for any unusual behaviors or obvious neurological symptoms (i.e. ataxia, convulsions, stereotypic behavior). Four days post ischemia the gerbils are sacrificed by decapitation and their brains removed and preserved in 10% buffered formalin. Brains were removed, fixed and stained with hematoxylin and eosin. Under a light microscope, hippocampal fields were observed and graded for damage to the CA1 subfield: 0 to 4 scale, with 0 representing no damage and 4 representing extensive damage.
Transient Focal Ischemia in Rats
The method was as described by Lin, T-N., He, Y. Y., Wu, G., Khan, M. And Hsu, C. Y. Effect of brain edema on infarct volume in a focal model cerebral ischemia model in rats. Stroke 24:117-121, 1993, which model is considered to be relevant to the clinical situation. Male Long-Evans rats 250-350 g were used. Surgery leading to focal ischemia was conducted under anesthesia with 100 mg/kg ketamine and 5 mg/kg i.m. xylazine. Rectal temperature was monitored and maintained at 37.0xc2x10.5 deg C. The right middle cerebral artery (MCA) was exposed using microsurgical techniques. The MCA trunk was ligated immediately above the rhinal fissure with 10-0 suture. Complete interruption of blood flow was confirmed under an operating microscope. Both common carotid arteries were then occluded using nontraumatic aneurysm clips. After a predetermined duration of ischemia (45 min), blood flow was restored in all three arteries. Twenty-four hours post occlusion, rats were killed under ketamine anesthesia by intracardiac perfusion with 200 ml of 0.9% NaCl. The brain was removed and processed with 2% triphenyltetrazolium chloride to identify and quantitate the infarcted brain region. Compounds were administered by intravenous infusion for 4 hours.